Protein delivery system using human papillomavirus virus-like particles

ABSTRACT

Human Papillomavirus virus like particles (VLPs) have been constructed so that they contain a modified L2 protein. The L2 protein has been minimized and is fused to a second protein or peptide. The fused protein is incorporated into the VLP and the VLP can deliver the protein to a cell. The modified VLPs can be used to increase the breadth of immune response in vaccine preparations or to deliver other proteins of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US99/17931, filed Aug. 10, 1999, whichclaims the benefit of U.S. provisional application 60/096,638, filedAug. 14, 1998, which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention is related to a method of delivering protein to a cell byfusing it to a modified papillomavirus L2 protein, and expressing thefused protein in a virus-like particle. The invention is also directedto modified genes encoding the proteins, the modified virus-likeparticles and host cells containing the virus-like particles.

BACKGROUND OF THE INVENTION

Human Papillomaviruses (HPVs) infect the genital tract and have beenassociated with various cancers and other diseases. Recombinantlyproduced L1 protein and L1+L2 protein can self-assemble to formvirus-like particles (VLPs). These VLPs are immunogenic, and may be thebasis of a vaccine.

Currently, however there is no known prophylactic vaccine or idealtherapeutic treatment of disease caused by HPV. An immune response toother HPV proteins co-delivered within the context of VLPs such as HPVearly proteins or genes may enhance the protective effect of a vaccine.Recently, a paper by Greenstone et al described VLPs in which the L2protein was modified to create a fusion protein between full length L2and HPV E2 or E7. (Greenstone et al, 1998 Proc. Natl. Acad. Sci. USA95:1800–1805.) The fusion proteins co-assembled with L1 into VLPs whichappeared indistinguishable morphologically from L1 VLPs and in theirability to elicit neutralizing antibodies. Furthermore, in tumorchallenge models, mice vaccinated with chimeric L1+L2: E7 fusion VLPswere found to protect mice from E7-expressing tumors.

However, there are potential problems associated with the use of afull-length L2 as a fusion partner. First, due to size retraints, anentire L2 as a fusion partner has a limited capacity to accept largefusion proteins. For instance, fusion of E1 with intact L2 of CRPV leadsto morphologically aberrant VLPs. Furthermore, expression of afull-length L2 will most likely affect the activity of enzymaticallyactive proteins and therefore may limit its usefulness to deliverfunctional proteins into a cell. The minimal sequence requirements of L2enabling coassembly with L1 into VLPs are unknown and the ability todelete significant portions of L2 while retaining the capacity toassemble with L1 is also unknown.

It would be desirable to provide a minimal L2 fusion protein which stillretains the protein delivery function of a full-length L2, yet candeliver larger and functionally-active proteins.

DESCRIPTION OF THE INVENTION

This invention is directed to a method of delivering a peptide orprotein to a cell comprising the steps of: a) fusing a nucleic acidsequence encoding the peptide or protein to be delivered to a modifiedpapillomavirus (PV) L2 gene to create a fusion protein gene, wherein theL2 gene is less than full-length and comprises at least the codingsequences for the amino-terminal 69 amino acids and the carboxy-terminal84 amino acids (aa) of L2; b) expressing the fusion protein gene in ahost cell to obtain a fusion protein; c) contacting the fusion proteinwith PV L1 protein under conditions wherein the fusion protein and theL1 protein spontaneously combine to form a virus-like particle (VLP);and d) delivering the VLP to a cell.

This invention also relates to nucleic acids encoding a fusion proteincomprising a less than full-length L2 protein fused to a gene encoding apeptide or protein of interest, and to these fusion proteins. Inparticular, this invention is related to L2 fusion proteins whichcomprise less than a full-length L2, and comprise at least the aminoterminal 69 amino acids and the carboxy terminal 84 amino acids of L2.Additionally, this invention relates to the virus-like particles (VLPs)which contain the fusion protein, and to cellular hosts containing theseVLPs.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the assembly of the minimal L2 DNA construct from nativeHPV16 L2. The coding sequence of HPV16 L2, shown in lower case italics,served as template for PCR amplifications. The initiator and terminatorcodons are in uppercase. Sense primers, I and A, are shown above thetemplate; anti-sense primers, C and D, below. New sequence information,introduced by PCR amplification are shown in uppercase. Thecomplimentary sequences in primers A and C are underscored.

FIG. 2 is the amino acid translation of the HPV 16 Minimal L2 Gene.Amino acid changes resulting from the introduction of the polylinkercontaining unique restriction sites Not I, Sac I, and Xho I areindicated by italics. Amino acids 1–69 are contributed from the aminoterminus of L2. A glutamic acid residue is added at position 70. Aminoacids 71–154 are contributed from the carboxyl terminus of L2. Atposition 72, a glutamic acid residue replaces a serine found in thefull-length wildtype L2.

FIG. 3 is a Western blot of immunoprecipitated yeast lysates, showingco-immunoprecipitation of L2-E chimeric proteins with HPV16 L1VLP-specific Mab H16: V5. The left panel shows anti-E1 stainedimmunoblot of minimal L2: E1 fusion (mL2: E1) clones E1-121 to E1-124,mL2: E2 sample E2-23 and no lysate samples are specificity controls. Themiddle panel shows anti-E7 stained immunoblot of minimal L2: E7 fusion(mL2: E7) clones E7-125 to E7-127 and E7-31; mL2: E2 sample E2-23 and nolysate samples are specificity controls. The right panelshows anti-E2stained immunoblot of mL2: E2 fusion clones E2-33 a, E2-33 b; sampleE2-33 b is also shown with no H16: V5 capture antibody as a control andYP3#1 no E protein control is shown as mL2 vector.

FIG. 4 is a table listing beta galactosidase activities of transientlytransfected cell lysates.

DETAILED DESCRIPTION OF THE INVENTION

The method of this invention can be used with virtually anypapillomavirus strain. In preferred embodiments, the HPV is one of thestrains which is associated with genital warts and/or genital cancers,and in particular may be selected from the group consisting of: HPV6 a,HPV6 b, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44,HPV45, HPV51, HPV52, and HPV56.

Wild-type virus-like particles are predominantly made of L1 protein,although they contain a minor amount of L2 protein. In accordance withthis invention, the VLPs have been modified so that they still containwild-type L1 protein, but now contain a deleted L2-fusion proteininstead of wild-type L2. The L2 segment of the fusion protein issubstantially less than the full length L2 protein; it contains peptidedomains from the amino terminus and from the carboxyl terminus which arenecessary for association with L1 proteins and incorporation into VLPs,at the least, for HPV type 16, the coding sequences for theamino-terminal 69 amino acids and the carboxy-terminal 84 amino acids(aa) of L2. The L2 sequence may be larger than this, as long as it isless than wild-type. Preferably the total amount of L2 amino acidspresent is less than about 60% of wild type, more preferably less thanabout 50% of wild-type, and even more preferably less than about 35% ofwild type. This modified L2 will be referred to throughout thespecification and claims as a “minimal L2” gene and protein.

Virtually any protein or peptide can be fused to the minimal L2 proteinto make the fusion protein. HPV proteins which are normally not part ofthe VLP are preferred if the goal is to make a VLP which induces anincreased immune response as compared to a wild-type VLP. For thisapplication, these proteins include but are not restricted to HPV: E1,E2, E3, E4, E5, E6, and/or E7, HIV TAT and/or beta lactamase.Particularly preferred proteins are E1, E2, and E7. These fusionproteins and the nucleic acids encoding them make up another aspect ofthis invention.

One of the advantages of this invention is that the fusion partner maybe a large protein. For example, the fusion partner may be over 50 kD,over 60 kD and even over 70 kD. This increased size allows for theintroduction of most known proteins, and makes this delivery systemparticularly useful not only for HPV-related proteins as describedabove, but also for proteins which are not associated treatment of withHPV disease.

Thus, this invention includes methods of using a modified VLP as ageneralized transporter, directing the delivery of virtually any desiredprotein or peptide into cells. In one embodiment, reporter or regulatoryproteins are delivered to cells as a measure of VLP uptake by cells. Asan example of fusion candidates which is by no means limiting, is theregulatory protein of HIV known as TAT. Fusions of TAT to the minimal L2have been shown to retain protein function as well as form VLPs with L1.This is useful in establishing neutralization assays.

The fusion protein and L1 protein may be made simultaneously bytransforming a selected host cell with genes encoding the proteins underconditions which allow expression of the two genes. General techniques,the genes for L1 s and L2, and methods for recombinant expression of L1and L2 proteins are known in the art, and may be used. Preferred hostcells include yeast, insect cells, mammalian cells and E. coli.

Under one preferred embodiment of this invention, yeast are transformedwith plasmid DNA containing the genes encoding L1 and the L2 fusionunder control of known yeast promoters, such as the yeast Gal 1 and Gal10 promoters. Expression of each gene product is induced by addition ofgalactose to the growth media, and the VLPs are isolated from theinduced cell lysates.

Alternatively, the one set of host cells may be transformed to expressonly L1 protein, and a second set of host cells may be transformed toexpress the L2 fusion protein of this invention. The respective proteinsare harvested, optionally subjected to a purification step, and arebrought into contact so that VLPs containing the fusion protein areformed.

The modified VLPs make up yet another aspect of this invention. TheseVLPs may be administered as a vaccine, or as part of a therapeuticregimen. Therapeutic or diagnostic compositions of the invention areadministered to an individual in amounts sufficient to treat or diagnosePV infections. The effective amount may vary according to a variety offactors such as the individual's condition, weight, gender and age.Other factors include the mode of administration. Generally, thecompositions will be administered in dosages ranging from about 1 mcg toabout 1 mg.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneously, topically, orally, mucosally,intravenously and intramuscularly.

The vaccines of the invention comprise DNA, RNA or proteins encoded bythe DNA that contain the amino-terminal and carboxyl-terminal portionsof L2 which permit the incorporation of proteins fused to it to beincorporated into VLPs. Such vaccines are also safe enough to beadministered without danger of clinical infection, do not have toxicside effects, can be administered by an effective route, are stable, andare compatible with vaccine carriers.

The vaccines may be administered by a variety of routes, such as orally,parenterally, subcutaneously, mucosally, intravenously orintramuscularly. The dosage administered may vary with the condition,sex, weight, and age of the individual; the route of administration; andthe type of PV of the vaccine. The vaccine may be used in dosage formssuch as capsules, suspensions, elixirs, or liquid solutions. The vaccinemay be formulated with an immunologically acceptable carriers oradjuvants, or other excipients.

The vaccines are administered in therapeutically effective amounts, thatis, in amounts sufficient to generate a immunologically protectiveresponse. The therapeutically effective amount may vary according to thetype of PV. The vaccine may be administered in single or multiple doses.

The purified proteins of the present invention may be used in theformulation of immunogenic compositions. Such compositions, whenintroduced into a suitable host, are capable of inducing an immuneresponse in the host.

The following non-limiting Examples are presented to better illustratethe invention.

EXAMPLE 1 Construction of the Modified L2 Gene

The modified HPV16 L2 gene was constructed from the coding sequences forthe amino-terminal 69 amino acids and the carboxy-terminal 84 aminoacids (aa) of HPV16 L2 which were fused in frame by a syntheticpolylinker that introduced unique Not I, Sac I, and Xho I restrictionendonuclease recognition sites and resulted in the insertion of oneglutamic acid residue and the mutation of a serine residue to glutamicacid. (FIG. 1).

PCR primers (Midland Certified Reagents; Midland, Tex.) were designed toamplify L2 sequences from the native L2 gene contained within thevector, pGal110 (Hofmann, K, et al 1995 Virology 209:506–518) into whichthe genes encoding HPV16 L1+L2 were inserted. (See FIG. 1).

Primers I (5′-CTT CCC CCC GGG CAC AAA ACA AAA TGC-3′; SEQ. ID. NO. 1)and C (5′-CTC GAG CTC GCG GCC GCC TGT ACC CGA CCC-3′; SEQ. ID. NO. 2)amplified a 265 base pair (bp) sequence encoding the amino-terminal 69aa and 23 bp of upstream untranslated sequence including a Sma Irestriction enzyme site. Primer C modified and extended the L2 aminoterminal-encoding region and appended Not I, Sac I and Xho I restrictionenzyme sites downstream of the L2-encoding sequences.

Primers A (5′-GCG GCC GCG AGC TCG AGG GTT ATA TTC CTG CAA ATA CAA-3′;SEQ. ID. NO. 3), C and D (5′-CCC TCC AGA TCT CTA GGC AGC CAA AGA GAC ATCTG-3′ SEQ. ID. NO. 4) amplified a 285 bp sequence encoding thecarboxy-terminal 84 aa of L2 plus 6 bp which added a Bgl II restrictionenzyme site. Primer A also appended a 17 bp sequence containing Not I,Sac I, and Xho I sites upstream of the L2-encoding sequence.

The minimal L2 expression construction was assembled throughcomplementary sequences added by primers A and C. The isolated DNAproducts of the I/C and A/D amplification reactions above were both usedin a PCR reaction which included the I and D primers. To facilitate thejoining of the fragments through their 17 bp complementary sequence,three PCR cycles were performed with the annealing temperature at 37°C., followed by 15 cycles with the annealing temperature of 57° C. Theresulting amplification product was blunt-end ligated into pcrScript(Stratagene, LaJolla, Calif.) and transformed into XL-1 Blue MRF′ cells(Stratagene). Positive clones were identified by PCR using primers I andD, and confirmed by restriction digest analysis. The construction wasthen verified by automated sequence analysis (Perkin Elmer, Inc., FosterCity, Calif.).

Plasmid DNA from an appropriate isolate was then digested with Sma I andBgl II; a fragment of approximately 0.5 kilobase pairs (kb) was gelpurified and ligated with the 14 kb Sma I and Bgl II vector fragment ofpGAL110 into which the HPV16 L1 gene was inserted adjacent to the GAL 1promoter. Competent DH5 E. coli cells (Gibco BRL, Rockville, Md.) weretransformed with the ligation mixture and transformants selected on LBampicillin plates (Remel, Lenexa, Kans.). Clones were initially screenedby PCR in which primers D and I were used to amplify portions of L2.Sequencing of candidate clone YP3#1 verified the sequence to be that asshown in FIG. 2.

YP3#1 was then employed as the backbone construct into which genesencoding HPV16 E1, E2, E7 or Tat open reading frames were inserted.

EXAMPLE 2 Insertion of HPV E Protein- and Tat-Encoding Genes

The gene encoding HPV16 E2 was obtained by PCR amplification of a HPV16positive clinical sample which was then inserted directly into thesubcloning vector pCRII (Stratagene) and sequence verified as above. TheE2 gene sequence was then modified in the following manner:

1) In-frame Xho I, Nae I, Not 1-containing DNA sequences were added tothe amino terminal portion of E2. Additionally, Not I, Nae I, and XhoI-containing sequences were added to the carboxyl-terminal portion of E2to facilitate insertion within E2 at the Not I, Xho I sites.

2) The DNA sequences were altered by PCR mutagenesis to encode alanineresidues encode at residues glutamic acid 39 and isoleucine 73. This wasdesigned to inactivate E2 protein function.

The modified HPV16 E2 gene described above was digested with Not I, XhoI and ligated with similarly digested YP3#1 vector. Transformantscontaining the properly-inserted E2 sequences were selected by PCRsequence verified and designated YP3-E2.

A similar approach was employed for inserting the genes encoding HPV16E1, HPV16 E7 and HIV Tat (creating YP3-E 1, YP3-E7 and YP3-Tat,respectively). The construct in which Tat was inserted at the carboxyterminus of minimal L2 was designated as YP3-cTat. For E1, glycine 482was altered to aspartic acid; for E7, cysteine 24 and glutamic acid 26were both changed to glycine to inactivate protein function. Theresultant constructions were then used to transform yeast.

EXAMPLE 3 Identification and Growth of Yeast Expressing Chimeric VLPs

Plasmid DNA of YP3#1 and derivatives described above were used totransform Saccharomyces cerevisiae (MATa, leu2-04, prb1:: HIS3, mnn9::URA3, cir^(o)) by the spheroplast method (Hinnen et al., 1978, Proc.Natl. Acad. Sci. USA 75:1929–1933). Transformed spheroplasts were platedonto selective (leucine minus) medium (Remel, Lenexa, Kans.). Cloneswere isolated through two rounds of single colony selection. Smallliquid cultures of candidate clones were grown to high cell density inmedium containing galactose. Crude extracts were prepared by vigorousagitation with glass beads followed by centrifugation. The clarifiedextracts were analyzed for expression of L1, the L2 component, and VLPsby various methods including SDS PAGE, ELISA, immunoblotting, and EIA,using monoclonal antibodies or monospecific polyclonal antisera thatrecognize L1, or L2, or the amino or carboxy termini of L2, or L1 VLPs,or E1, or E2, or E7, or any other protein or peptide fused to themodified L2. Clones which expressed the L2 component and formed VLPswere selected for further characterization. One-liter or 16-litercultures of selected clones were grown in galactose containing mediumfor large-scale preparation of chimeric VLPs.

EXAMPLE 4 Purification of HPV Type 16 L1/L2_(mini)/E2 Chimeric VLPs

Cell pellets from a 16-liter fermentation of S. cerivesiae were storedfrozen at −70° C. Frozen cells “Breaking Buffer” (200 mM MOPS, pH 7, 1mM CaCl₂) was added to give approximately 20% (w/v) slurry. BENZONASE®(Nycomed Pharma) was added to 750 units/g wet cell weight. The cellslurry was broken at a pressure of approximately 19,000 psi by 5 passesin a M110-Y Microfluidizer (Microfluidics Corp., Newton, Mass.). Thecell lysate was clarified by microfiltration through a 0.65 micron poresize hollow-fiber cartridge and then diafiltered with three volumes of0.25 M sodium citrate, 0.2 M MOPS, pH 7.0. Permeate was loaded onto aPOROS® 50 HS resin (Perseptive Biosystems, Cambridge, Mass.)equilibrated in 200 mM MOPS, pH 7, 250 mM sodium citrate. The column waswashed with 50 mM MOPS, 0.5 M NaCl, 5 mM sodium phosphate, pH 7 andeluted with a linear gradient from 0.5 to 1.5 M NaCl in the same buffer.Column fractions were analyzed by immunoblotting and SDS-PAGE withcolloidal Coomassie detection.

The 50 HS pool was filtered through a 0.22 mm filter and applied to aceramic hydroxyapatite (HA) Type II (Bio-Rad) column. The column waswashed with 50 mM MOPS, pH 7, 1.25 M NaCl, 5 mM sodium phosphate andeluted with a linear gradient from 5 to 200 mM sodium phosphate, pH 7 in1.25 M NaCl. Fractions were analyzed by Western blot and SDS-PAGE.Fractions showing comparable purity and enrichment of L1 protein werepooled. The pooled fractions were filtered aseptically through a 0.22 mmmembrane and submitted for electron microscopy.

EXAMPLE 5 Detection of VLPs by Electron Microscopy

Transmission electron microscopy was performed by EMBS (Elkridge, Md.).Appropriately diluted samples were placed on a 300 mesh carbon-coatedcopper grid and allowed to air dry. Grids were stained withphosphotungstic acid. All microscopy was performed using a JEOL 1200 EXtransmission electron microscope. The micrographs generated had a finalmagnification of 138,000×. Electron microscopy confirmed the presence ofintact VLP particles with a mean diameter of 32 nm. These particles wereindistinguishable morphologically from yeast-expressed L1 or L1+L2particles. Confirmation of the presence of L2 fusions within the VLPswas demonstrated by immunoblotting analysis of process fractions (SeeExample 6).

EXAMPLE 6 Co-Immunoprecipitation of L2 Fusion Proteins with L1 SpecificAnti-VLP Antibodies

To show that the L2 fusions were an integral component of the VLPs, VLPswere captured by an HPV16 L1 VLP-specific monoclonal antibody (mAb) H16:V5 (Wang, D. et al. 1997 J. General Virology, 78: 2209–2215), washedextensively and the retained products detected by immunoblotting. Inbrief, magnetic beads precoated with sheep anti-mouse IgG (DynabeadsM-280; Dynal, Oslo) were washed with TMOPS buffer (0.05M MOPS, pH 7.0,0.4M NaCl, 0.1% Tween-80) containing 5% non-fat dry milk. The washedbeads were incubated overnight at 4° C. with H16: V5 mAb in the samesolution. Unbound mAb was removed by washing with TMOPS. Beads were thenincubated with control or VLP-containing crude yeast extracts (seeExample 3) or partially-purified VLP preparations (see Example 4).Extracts containing 100–400 μg total protein were diluted with 0.2 MOPS,pH 7.0, 2 mM MgCl₂ and incubated with beads for 4 hours at 4° C. Beadswere then washed extensively with TMOPS; bound proteins were recoveredby heating the beads at 95° C. in Laemmli sample buffer, resolved bySDS-PAGE and identified by immunoblotting.

Identification of the L2 fusion protein was performed with antibodiesthat recognize the L2 portion of the protein and by antibodies thatrecognize the protein of interest fused to L2, for example E1, E2, orE7. In addition to the inclusion of control yeast extracts, othercontrols included 1) beads which have not been incubated with H16: V5,and 2) beads which have not been incubated with yeast extract. FIG. 3shows the immunoblot results of VLPs captured by the H16: V5 Mab andimmunoblotted as described above and then stained with anti-E1, anti-E7or anti-E2 antisera. In each case for multiple isolates, the E-protein:L2 fusions were detected amount the captured, purified VLPs using theappropriate antibody, but not in the absence of lysate or using purifiedchimeric VLPs containing E-proteins different from the detectionantisera.

Coprecipitation of the L2 fusion protein with L1 using a monoclonalantibody specific for L1 VLPs was taken as an indication that the L2fusion protein was associated with the VLP.

EXAMPLE 7 Demonstration of Tat Activity in Tat/Minimal L2 Fusions inMammalian Cells

To demonstrate that Tat/L2 fusions retained Tat activity, an expressionplasmid was constructed in which DNA encoding the HIV Tat gene wasinserted downstream of the minimal L2 gene so that it would be linked tothe carboxyl terminus of L2 in the translated product. This DNA cassettewas digested with Bgl II and inserted within the Bgl II site of themammalian expression vector, V1 Jp (Montgomery, D. et al 1993 DNA & CellBiol. 12:777) adjacent to the CMV promoter. The L2/Tat fusion wascreated by PCR of the minimal L2 (lacking a translational stop signal)using the oligomers 5′-TCC CCC GGG AGA TCT GCC ACC ATG CGA CAC AAA CGTTCT GCA AAA C-3′ (primer W; SEQ ID NO:5) and 5′-GGC AGC CAA AGA GAC ATCTG-3′ (primer X; SEQ ID NO:6). Oligomers 5′-CAG ATG TCT CTT TGG CTG CCATGG AGC CAG TAG ATC CTA GAC-3′(primer Y; SEQ ID NO:7) and 5′-CTC GTA AGATCT CTA TTC CTT CGG GCC TGT C-3′(primer Z; SEQ ID NO:8) were used toamplify the complete Tat open reading frame and to introduce sequencecomplementarity with L2. The products from both PCR reactions wereagarose gel isolated and then combined in a second PCR reaction in whichonly primers W and Z were added which generated the L2/Tat fusionthrough overlap of complementary sequences. These primers also appendedBgl II sites which were digested to facilitate insertion within V1 Jp toform V1 Jp-L2 cTat.

Activity of the L2/Tat fusion was determined by transfection of V1 Jp-L2cTat into P4 R5 cells using a calcium phosphate transfection kit(available from Gibco BRL, Rockville, Md.) performed as recommended bythe manufacturer. P4 R5 cells contain an integrated copy of the geneencoding beta galactosidase under control of the HIV LTR. If theintroduced Tat gene encodes functionally active Tat, expressed Tatprotein will bind the LTR and result in expression of betagalactosidase. Lysates were made of 48 hr post transfection cultures andbeta galactosidase activity assessed using Galactostar® reagents(Tropix, Bedford, Miss.). The results are summarized in FIG. 4 whichshows that there were similar amounts of beta galactosidase produced incells transfected with V1 Jp-L2 cTat as with the unfused positivecontrol pD5-Tat plasmid (235 vs.

175). The no-Tat DNA control had a value of 0.462. Thus, full Tatactivity appears to be retained within the L2/Tat fusion.

1. A method of delivering a peptide or protein to a cell comprising: a)fusing a nucleic acid sequence encoding the peptide or protein to amodified human papillomavirus (HPV) L2 gene to create a fusion proteingene; b) expressing the fusion protein gene in a host cell to obtainfusion protein; c) contacting the fusion protein with HPV L1 proteinunder conditions wherein the fusion protein and the L1 proteinspontaneously combine to form a virus like particle (VLP); and d)delivering the VLP to the cell; (i) wherein the modified HPV L2 geneencodes less than about 50% of wild type L2 protein and encodes domainsfrom the amino and the carboxyl termini; and (ii) wherein the HPV L2gene is from an HPV strain selected from the group consisting of: HPV6a,HPV6b, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44,HPV45, HPV51, HPV52, and HPV56.
 2. A HPV virus-like particle (VLP)comprising HPV L1 protein and a fusion protein wherein the fusionprotein comprises: a first protein which is an HPV L2 protein which isless than about 50% of wild type; and a second protein or peptide.
 3. AVLP according to claim 2 wherein the HPV L2 protein is from a HPV strainselected from the group consisting of: HPV6 a, HPV6 b, HPV11, HPV16,HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52,and HPV56.
 4. A host cell comprising the VLP of claim
 2. 5. A nucleicacid encoding a VLP according to claim 3.